The Hippo Pathway in Colorectal Cancer

FOLIA HISTOCHEMICA Review ET CYTOBIOLOGICA Vol. 53, No. 2, 2015 pp. 105–119

The Hippo pathway in colorectal cancer

Piotr M. Wierzbicki, Agnieszka Rybarczyk

Department of Histology, Faculty of Medicine, Medical University of Gdansk, Gdansk, Poland

Abstract Colorectal cancer (CRC) is one the most frequently diagnosed neoplastic diseases worldwide. Currently, aside from traditional chemotherapy, advanced CRCs are treated with modern drugs targeting cellular components such as epithelial growth factor receptor (EGFR). Since up to 70% of metastasized CRCs are drug resistant, the description of recent progress in cellular homeostasis regulation may shed new light on the development of new molecular targets in cancer treatment. The Hippo pathway has recently become subject of intense investigations since it plays a crucial role in cell proliferation, differentiation, apoptosis and tumourigenesis. Components of the Hippo pathway are deregulated in various human malignancies, and expression levels of its major signal transducers were proposed as prognostic factors in colorectal cancer. In this review we focused on recent data regarding Hippo pathway, its up-stream signals and down-stream effectors. Hippo negatively regulates its major effectors, YAP1 and TAZ kinases, which act as transcriptional co-activators inducing expression of genes involved not only in tissue repair and proliferation but are also oncoproteins involved in tumour development and progression. The deregulation of Hippo pathway components was found in many malignancies. The interactions between Hippo and Wnt/b-catenin signalling, crucial in the maintenance of cell homeostasis, have been described in relation to the control of intestinal stem cell proliferation and CRC development. The recently discovered positive feedback loop between activated YAP1 and increased EGFR/KRAS signalling found in oesophageal, ovarian and hepatocellular cancer has been related to the CRC progression and resistance to EGFR inhibitors during CRC therapy. (Folia Histochemica et Cytobiologica 2015, Vol. 53, No. 2, 105–119)

Key words: CRC; Hippo pathway; YAP1/TAZ; Wnt signalling; EGFR/KRAS; intestinal stem cells

Abbreviations: AJ — adherent junction, AMOT — tumor suppressor kinase 1/2, Lgr5 — leucine-rich angiomotin, APC — adenomatous polyposis coli, repeat-containing GPCR5, LPR — Lipoprotein AREG — amphiregulin, Axl — Axl receptor tyrosine receptor-related protein, MOB1 — MOB kinase kinase, BMI1 — BMI1 polycomb ring finger proto-on- activator 1, MST1/2 — serine/threonine kinase 4/3, cogene, β-TrCP — beta-transducin repeat containing NF2 — neurofibromin 2, Oct-4 — octamer-binding E3 ubiquitin protein ligase, CK1 — casein kinase 1, transcription factor 4, RASSF — Ras association CRC — colorectal cancer, CTGF — connective tissue (RalGDS/AF-6) family member, SAV1 — Salvador growth factor, DSS — dextran sodium sulphate, DVL family WW domain containing protein 1, STRIPAK — Dishevelled protein, ECM — extracellular matrix, — striatin interacting phosphate and kinase complex, EGFR — epithelial growth factor receptor, EMT — TAZ — tafazzin, TCF4 — transcription factor 4, epithelial-to-mesenchymal transition, FAT — FAT TEAD — TEA domain family member, TJ — tight atypical cadherin, FZD — Frizzled protein, GPCR junction, b-TrCP — b-transducin repeat-containing — G protein coupled receptor, GSK3 — glycogen protein, VEGF — vascular endothelial growth factor, synthase 3, ISC — intestinal stem cell, KO — knock WB — Western blot technique, YAP1 — Yes-asso- out, KRAS — Kirsten rat sarcoma, LATS1/2 — large ciated protein 1

Colorectal cancer Correspondence address: P.M. Wierzbicki, Ph.D. Department of Histology, Faculty of Medicine Medical University of Gdansk Colorectal cancer (CRC) is the third most commonly Debinki St. 1, 80–211 Gdansk diagnosed cancer in males and the second in females, tel.:+48 58 349 15 01, +48 58 349 14 37, fax: +48 58 349 14 19 with an estimated 1.4 million cases and 693,900 deaths e-mail: [email protected] occurring in 2012 [1]. Five-year relative survival ranges

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Figure 1. The mammalian Hippo pathway. Drosophila orthologue genes are shown below the names of the mammalian proteins. When the Hippo pathway is not active (e.g. during cell injury and repair or in cancer), the active effector proteins, YAP1 and TAZ, interact with TEAD1-4 transcription factors and promote transcription of genes involved in cell proliferation. During normal cell homeostasis, when the Hippo pathway is active, YAP1 or TAZ are inhibited due to their phosphorylation by core components of the Hippo pathway (SAV1, MST 1/2, LATS1/2 — shown in the central rectan- gle). In a phosphorylated form cytoplasmic YAP1/TAZ may interact with (i) the 14-3-3 protein, (ii) components of cell junctional complexes like AMOT or b-catenin, or (iii) may be degraded in proteasomes. YAP1/TAZ can be also regulated by other mechanisms such as cell polarity, GPCR signalling or ECM stiffness as described in the body text. Acronyms are explained on the first page from more than 90% in patients with stage I disease tics and so called biological drugs [6]. In this review to about 10% in patients with stage IV disease [2]. we also aimed to bring closer the possible associations Treatment of CRC is one of the most expensive when between the Hippo pathway and drug resistance of diagnosed at later stages when prognosis is generally CRC cells. poor [3]. Although the knowledge of the background and the development of CRC has recently increased, The Hippo pathway there is a high need for more studies to found out well working prognostic, survival and diagnostic markers. The Hippo pathway is an important regulator of cell Serum markers for routine CRC diagnostic such as CEA proliferation, growth and apoptosis [7, 8]. Moreover, (carcinoembryonic antigen) and CA 19-9 showed good it controls tissue homeostasis, organ size and stem cell prognostic values and have been used as CRC tumour functions. Its deregulation is frequently observed in predictors [4]. The aim of this paper was to provide many human cancers, suggesting that alterations of rationale to consider the expression of Hippo pathway Hippo signalling are connected with tumour initia- genes as possible new prognostic genes in CRC. tion and/or progression [9–11]. Hyperactivation of The modern chemotherapy has adapted molecu- the Hippo pathway downstream effectors — YAP1 lar findings in many cancers to focus on activation/ (Yes-associated protein 1) and TAZ (transcriptional /inactivation of cancer-related intracellular pathways co-activator with PDZ binding motif) may contribute to eliminate or decrease expansion of tumour cells. to the development of cancer, however, their activa- Such drugs which target vascular endothelial growth tion may also play a positive role in stimulating tissue factor-A (VEGF, Bevacizumab) or epidermal growth repair and regeneration following injury [12, 13]. factor receptor (EGFR, Cetuximab and Panitumumab) The general scheme of the Hippo pathway and inter- have been introduced for the treatment of CRC [5]. actions of mammalian Hippo components with up- However, it was noted that 50–70% of advanced CRC stream signals and effectors are presented in Figure 1 cases were resistant to both classical chemotherapeu- and listed in Table 1.

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Table 1. Names and main functions of the most important Hippo pathway genes and proteins

Mammalian gene Drosophila gene, protein Protein name, aliases Protein function NF2 Mer, Merlin Neurofibromin 2, Merlin, ACN, Signal transduction between cell-membrane SCH, BANF and cytoskeletal proteins WWC1 Kibra, ortholog WW and C2 domain containing 1, Phosphoprotein involved in cell polarity, KIBRA, HBEBP3, HBEBP36 mitosis and cell migration [105] FRMD6 Ex, Expanded FERM domain containing 6, EX1, Protein linking cytoskeleton with plasma Willin membrane [106] STK4 Hpo, Hippo Serine/threonine kinase 4, Cytoplasmic kinase involved in stress- MST1, KRS2 -induced mitogen-activated protein kinase cascade STK3 Serine/threonine kinase 3, Kinase activated by proapoptotic molecules MST2, KRS1 for the promotion of chromatin condensation during apoptosis, cardiomyocyte proliferation [107], regulation of osteoblast/osteoclast differentiation [108] RASSF1 Ras association (RalGDS/AF-6) Signal transducer, microtubule stabilization, domain family member 1, cell cycle arrest [109] RASSF1A, NORE2A SAV1 Sav, Salvador Salvador family WW domain Regulation of protein degradation, RNA containing protein 1, SAV, WW45 splicing and DNA transcription LATS1 Wts, Warts Large tumour suppressor kinase 1, Serine/threonine kinase involved in mitosis: WARTS interacts with CDC2/cyclin A LATS2 Large tumour suppressor kinase 2, Kinase which interacts with centrosomal pro- KPM teins aurora-a and ajuba during mitosis [110] MOB1A Mats MOB kinase activator 1A, MOB1, Protein involved in mitotic exit network [111], MATS, microtubule stability control [112] YAP1 Yki Yes-associated protein 1, YAP, YKI Downstream effector of Hippo pathway involved in development, repair and homeostasis TAZ Tafazzin, EFE, Taz1 Non-specific phospholipid-lysophospholipid transacylase involved in cardiolipin turnover [113]

Discovery and function of the Hippo pathway by Wts kinase, which is associated with an activating in Drosophila subunit Mats [19]. The kinase activity of Hpo could be antagonised by dSTRIPAK — phosphatase com- The Hippo pathway was first described almost 20 year plex [20]. The core kinase cassette of the Hippo pathway ago as a result of screening for mutant tumour suppres- acts as a suppressor of a downstream element — Yorkie sors in Drosophila. It was found that loss-of-function (Yki) [21]. Subsequent biochemical studies showed mutation of the Hippo pathway components revealed that Wts directly phosphorylates and inhibits Yki. Yki enormous overgrowth of fruit flies as a result of in- is a transcriptional co-activator that lacks DNA bind- creased cell proliferation and decreased apoptosis [14]. ing domain. It cooperates with nuclear transcription The first identified elements of the pathway were its factors, like Scalloped, and enables transcription of core components: warts (wts), hippo (hpo) and salvador genes which promote cell proliferation (e.g., Myc) and (sav) (Figure 1) [14–17]. These genes belong to the inhibits apoptosis (e.g., diap1) [22, 23]. When the cell hyperplastic group of Drosophila tumour suppressors, receives growth inhibiting signals, i.e. due to contact wherein mutations of these genes result in robust tissue inhibition, it activates core components of the pathway overgrowth without alterations of cell differentiation that phosphorylate Yki. This leads to the cytoplasmic status or cell polarity [18]. Further studies indicated sequestration of Yki and its binding to the 14-3-3 pro- that Hpo kinase in association with its adaptor protein tein, which finally results in Yki protein degradation Sav, phosphorylates and activates complex formed [21, 24].

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The components and function of the Hippo pathway the Hippo pathway in a cooperative manner or via in mammals direct binding of Ex and Yki to restrain Yki level in cytoplasm and inhibit its transcriptional activity [35, 36]. The core kinases cassette and downstream effectors of Function of this apical protein complex is con- the Drosophila Hippo pathway are highly conserved in served also in mammals. The complex of Nf2 (neurofi- mammals (Figure 1): MST 1/2 (homologues of Hpo), bromin2, a Mer ortholog), KIBRA and FRMD6 (also SAV1 (Sav homologue), LATS 1/2 (Wts homologues), known as WILLIN, as potential Ex ortholog) inhibits MOB1 (homologues of Mats) and YAP1 and its para YAP1 activity (Figure 1) [37]. Nf2, a tumour sup- logue TAZ (homologues of Yki) [25]. It was shown that pressor gene is currently the only one known Hippo expression of human genes can rescue the phenotypes pathway gene that is mutated in cancer (Table 2) [10], of corresponding Drosophila mutants in vivo [15, 19, 21]. especially in cancers of central nervous system [38]. The first kinase MST1/2 can be activated in two dif- Planar cell polarity not only regulates the position ferent ways: (1) by caspase-dependent cleavage under of a cell in the epithelial layer, but also plays a crucial apoptotic stress [26], or (2) by binding to one of Ras as- role in the control of development. In Drosophila the sociation domain family (RASSF) proteins, RASSF1A protocadherins Fat (Ft) and Dachsous (Ds) modulate [27]. Activated MST1/2 interacts with SAV1 through Wts activity; however, the role of their mammalian the SARAH domains presented on both proteins orthologues in the Hippo pathway is not so well un- what leads to the phosphorylation and activation of derstood and requires further studies [39]. their direct substrates LATS1/2 [28]. MOB1 protein Tight junctions (TJ) and adherens junctions is also phosphorylated by MST1/2, which results in its (AJ) play an important role in intercellular con- enhanced interaction with LATS1/2 and formation of tacts being responsible for the permeability barri- a complex that phosphorylates and inhibits activity of ers. Structural proteins of TJ and AJ were shown YAP1 and/or TAZ (Figure 1) [28, 29]. to interact with YAP1 (Figure 1). Cytoplasmic LATS1/2-dependent phosphorylation of Ser127 angiomotin (AMOT) proteins can inhibit YAP1/ of YAP1 and Ser89 of TAZ in sequences HXRXXS /TAZ via physical interactions and transfer of (H-histidine; R-arginine; S-serine; X-any amino acid) the YAP1/AMOT complexes from cytoplasm to are the most important reactions within this mecha- tight junctions or actin cytoskeleton (Figure 1). nism since this results in binding of 14-3-3 sites and causes Moreover, AMOT proteins also activate LATS1/2 segregation of YAP1 and TAZ in the cytoplasm [30–32]. to phosphorylate YAP [40]. Another TJ protein, When LATS1/2 phosphorylates Ser397 of YAP1 and ZO-2, was reported to increase nuclear localiza- Ser311 of TAZ, casein kinase1e/d subsequently phos- tion of YAP and tight junction localization of phorylates Ser400 and Ser403 of YAP1 and Ser314 of TAZ [41, 42]. It was demonstrated that a-catenin, TAZ thereby leading to ubiquitination and degradation a component of adherens junction, together with of YAP1 and TAZ proteins [31]. However, it has to be 14-3-3 protein and phosphorylated form of YAP1 noted that many other kinases, not involved in the Hip- form a complex that inhibits YAP1 activity [43]. po pathway, can phosphorylate YAP1 in a specific way, It has been shown that YAP1/TAZ activity can depending on the signals received by the cell. Akt kinase, also depend on environmental cues such as extracel- a pro-survival kinase, was shown to negatively regulate lular matrix stiffness, cell tension, cell attachment/ the YAP1-dependent transcription of pro-apoptotic /detachment [38, 44]. It was observed, that the nuclear genes [31]. It was also found that after DNA damage, localization of YAP/TAZ resulting in TEAD genes’ c-Abl kinase phosphorylates YAP1 at Y407 (tyrosine activation was found during proliferation of endothe- phosphorylation) which enhances the YAP1-p73 inter- lial and epithelial cells as well as during osteoblast action, prevents Itch-mediated ubiquitination of p73 and differentiation [38]. In breast cancer (MCF10A) and activates transcription of pro-apoptotic genes [33, 34]. mouse fibroblast NIH-3T3 cell cultures it was found that YAP1 overexpression and phosphorylation can Regulation of the YAP1/TAZ activity overcome cell contact inhibition [29]. Recent reports appraise the role of G protein-coupled receptors Upstream regulators of YAP1/TAZ activity can act (GPCRs) and their cooperation with Rho GTPase either through Hippo pathway core components or and actin cytoskeleton in negative or positive modi independently of Hippo kinases. For instance, in the fications of YAP1 or TAZ activity. Depending on epithelial cells apical-basal polarity and planar cell the class of G proteins, but also on the type of their polarity regulate the Hippo pathway. In Drosophila ligands, the effects on YAP1/TAZ activity regulation epithelium apically localized Merlin (Mer), Expanded might be completely different. For instance, lysophos- (Ex) and Kibra proteins form a complex (Figure 1) phatidic acid, sphingosine 1-phosphate and peptide which acts by binding to Sav, Hpo and Wts to activate agonists of thrombin receptors induce, whereas

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Table 2. The involvement of Hippo pathway proteins in human diseases

Gene’s name Role in human diseases Role in CRC development NF2, Merlin Mutation leads to neurofibromatosis type II; Barely studied; point mutations were not critical [49] lesions in the CNS and eyes and skin [114, 115] TSG in some cancers due to mutation and underexpression [10] WWC1, KIBRA Polymorphisms associated with Alzheimer’s Not studied disease [116] FRMD6, human Polymorphisms associated with Alzheimer’s disease Not studied Expanded [117], CCL: downregulation leads to resistance to Taxol I MC10FA cells [118] STK4, MST1 and Phosphorylation → loss of function in prostate cancer Decreased mRNA level in tumour associated with STK3, MST2 [119], CCL: overexpression inhibits cell proliferation node metastasis [121], mouse double KO: crypt and promote apoptosis in HepG2 cells [87] dysplasia, colon adenoma [46, 78, 120] Mouse double KO: cholangiocarcinoma [46, 101], HCC [120] RASSF1, RASSF1A TSG: promoter hypermethylation and decrease TSG; promoter hypermethylation and decrease expression in cancer: thyroid [122], oesophageal [123], expression in CRC [126, 127] prostate [124], colorectal, breast [125] SAV1, Salvador TSG: LOH and downregulation in renal cell carcino- No gene mutation in CRC [129] ma [128], no gene mutation in stomach, liver and lung cancer [129] CCL: overexpression induces apoptosis in MCF-7 cells [130] LATS1 TSG: decrease expression in cancer: glioma [131], TSG: promoter hypermethylation and mRNA NSLC [132], sarcoma [133] and astrocytoma [134]. decrease in CRC [86] CCL: LATS1 degradation inhibits apoptosis in MCF10A cells [135] LATS2 TSG: decrease expression in: malignant mesothelioma TSG: downregulation in CRC [87] (and LOH) [136], NSLC (no mutation found) [137] and astrocytoma [134] OG: overexpression in AML [138], nosopharyngeal carcinoma [139] MOB1, Mob1 Targeted by NS5A protein of hepatitis C virus [140]. TSG: downregulation in CRC [142] Mouse KO: various cancer types developed [141] TAZ Mutations in Barth syndrome [143] OG: overexpression in CRC [85] OG: overexpression in breast cancer [144], HCC [145] YAP1 OG: overexpression in NSCLC [88], prostate [90], OG: overexpression in CRC [83], in CRC cases breast [91], gallbladder cancer [146] and glioma [89] resistant to cetuximab [102] TSG: decreased expression in breast cancer [92] TSG: underexpression in CRC [79]

AML — acute myeloid leukaemia; CCL — cancer cell line(s); HCC — hepatocellular carcinoma; KO — knock out; LOH — loss of heterozygosity; NSCLC — non-small cell lung cancer; OG — oncogene; TSG — tumour suppressor gene epinephrine or glucagon repress YAP1/TAZ activity actions within the Hippo pathway. Moreover, cross [45, 46]. YAP1 could also be regulated by matrix talks of the Hippo pathway with other signalling stiffness independently from LATS1/2 phosphoryla pathways like Notch, EGF or Sonic hedgehog have tion. In cells cultured in soft matrix YAP and TAZ also been described [49]. Due to the space con- become phosphorylated even if all known LATS1/2 straints they will not be discussed. However, in this phosphorylation-dependent sites in YAP protein are review we focus on the relationships between Hippo inactivated by mutations [47, 48]. and Wnt-b catenin signalling pathways because of their crucial role for the maintenance of intestinal Hippo pathway cross talk with Wnt/b-catenin epithelium homeostasis (Figure 2A). Additionally, and EGFR/KRAS signalling we will also discuss cross talk between Hippo and EGFR/KRAS signalling pathways since their inter- The above presented ways of YAP1/TAZ activity actions may be involved in drug resistance during regulation show the complexity of molecular inter- CRC chemotherapy (Figure 3) [50, 51].

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Figure 2. Interactions between Hippo pathway effectors TAZ and YAP and Wnt/b-catenin pathway. A. When Wnt pathway is not triggered (WNT OFF), b-catenin and TAZ are inactivated by degradation via destruction complex. When Wnt is triggered (WNT ON), e.g. during tissue regeneration, the destruction complex is bound to Wnt/Frizzled and LRP5/6 resulting in the inhibition of either b-catenin or TAZ degradation. This leads to the accumulation of b-catenin and/or TAZ in the nucleus [38, 57–59]; B. Wnt/b-catenin pathway is restricted by TAZ inhibition. On the contrary, YAP1 gene expression is enhanced by b-catenin/TCF4 complex [60]

Figure 3. Indirect relationship between EGFR/KRAS and YAP. A positive autocrine regulation between EGFR and YAP1 is established via amphiregulin expression [50, 61]. Cetuximab and Panitumumab are humanized monoclonal antibodies against EGFR used in CRC therapy [61]

The interactions of the Hippo adhesion, cell polarity, growth factors or other small and Wnt signalling pathways signalling molecules, like hormones and cytokines, control whether a cell should enter process of prolif- Organs grow up to pre-defined size and shape, which eration, apoptosis or differentiation. Several studies are controlled by different signalling pathways that have emphasized the critical role of the Wnt/b-catenin receive external signals and translate them into re- pathway for gastrointestinal (GI) tract homeostasis. spective cellular processes. Cell-cell junctions, cell Uncovering its interactions with Hippo pathway

©Polish Society for Histochemistry and Cytochemistry Folia Histochem Cytobiol. 2015 www.fhc.viamedica.pl 10.5603/FHC.a2015.0015 Hippo pathway in colorectal cancer 111 increased our understanding of the mechanisms of Hippo upregulates EGFR/KRAS pathway intestinal epithelium homeostasis in physiology and pathology [38]. Recent reports [50, 61–63] revealed that the Hippo The effector of the Wnt signalling (WNTS) path- pathway effector, YAP1 protein, is involved in the way is the transcriptional co-activator b-catenin [52]. regulation of EGFR/KRAS signalling (Figure 3). Dur- WNTS ligands include many types of proteins that act ing homeostasis, degradation of YAP1 is triggered by as morphogens, controlling cell differentiation and ubiquitin ligase complex substrate recognition factors proliferation. These molecules bind to cell membrane SOCS5/6. It was found that during cancerogenesis receptor complexes composed of Frizzled (FZD) and EGFR-activated Ras, a small GTP-binding protein, Lipoprotein receptor-related (LPR) proteins and downregulates SOCS5/6 expression, which increases thereby regulate the activity of b-catenin [38, 53, 54]. level and half time of YAP1 in the cytoplasm [50]. b-catenin plays a central role in several developmental Non-phosphorylated YAP1 may induce transcription processes, such as regulation of gene transcription of EGFR as well as AREG (amphiregulin) genes. Ex- [53], stem cell renewal [55] and epithelial-to-mes- pression of the EGFR gene leads to up-regulation of enchymal transition (EMT) [56]. When a cell does the above-described positive feedback loop (Figure 3). not receive WNT signals, b-catenin is maintained at Moreover, amphiregulin proteins (secretory products low level in the cytoplasm through its degradation of AREG gene) bind to EGFR and act as autocrine in AXIN destruction complex. This complex is com- growth factors. Such relationship between YAP1 and posed of the scaffolding protein AXIN, the tumour the EGFR pathway was found to be important in the suppressor adenomatous polyposis coli gene product progression of oesophageal cancer in patients treated (APC), casein kinase 1 (CK1) and glycogen synthase 3 with EGFR inhibitors [61]. Recently published results (GSK3) [38, 53, 57, 58]. Due to sequential CK1 and of knockdown of YAP1 and knockdown of ERBB3 GSK3 phosphorylation of b-catenin N-terminal study suggested presence of an autocrine loop be- region, the phosphorylated b-catenin could be re tween YAP1 and EGFR which may control ovarian cognized by b-TrCP, an E3 ubiquitin ligase subunit cell tumourigenesis and cancer progression [63]. so that subsequent b-catenin ubiquitination leads to its proteasomal degradation (Figure 1 and 2A). The role of the Hippo pathway in intestinal Continuous elimination of b-catenin prevents it from epithelial cells nuclear translocation, and thereby Wnt target genes are repressed by the DNA-bound T cell factor/lym- Hippo pathway in intestinal epithelium homeostasis phoid enhancer factor, belonging to the TCF/LEF family of transcriptional factors [54]. The inner surface of the intestinal tube is lined with Binding of Wnt ligand induces the formation of a single layer of epithelial cells that play both absorbing a complex composed of FZD receptor, LRP5/6 co-re- and secreting functions. Major differentiated cell ceptor and WNT ligand together with the recruitment types are enterocytes (which are involved in the up- of the scaffolding protein Dishevelled (DVL) [54]. In take of nutrients), goblet cells (which produce mucus), effect, since b-catenin is not degraded, it accumulates enteroendocrine cells (which produce hormones) and in the cytoplasm and migrates to nucleus, where it Paneth cells. Except for Paneth cells, all cell types forms a complex with TCF/LEF what enables tran- migrate as clonal lineages to the tip of the villus within scription of Wnt target genes [38, 53, 59]. 4–5 days, where they are shed into the lumen [64]. In 2010 Varelas et al. found that activation of the Post-mitotic Paneth cells are relatively long-lived (5–6 Hippo pathway restricts Wnt/b-catenin signalling by in- weeks) [65] and intermingle with intestinal stem cells teraction of TAZ and DVL in the cytoplasm (Figure (ISC) at crypt bottoms to secrete ISC niche factors 2B) [60]. TAZ interaction with DVL inhibits CK1 and function in innate immunity. Colon has a simple binding and Wnt3A-induced DVL phosphorylation, columnar epithelium that lacks Paneth cells; instead, thereby inhibiting Wnt3A-induced transcriptional re- so called ‘deep secretory cells’ are thought to be response. Loss of TAZ in a cell culture and in the kidneys sponsible for the ISC niche function [64, 66]. Regard- of Taz-null mice results in increased DVL2 phosphoryla- less of the differences in morphology and functions, tion, enhanced cytoplasmic and nuclear b-catenin epithelial cell types in intestinal crypts are organized accumulation and Wnt-target genes’ induction [60]. into three compartments. All epithelial lining cells are Furthermore, the inhibition of the Hippo pathway derived from intestinal stem cells that occupy the base activity leads to increased nuclear TAZ level so that of crypts (1st compartment). ISC divide, migrate and reduced TAZ-DVL binding results in increased target start to differentiate into transient-amplifying cells gene expression in both pathways (Figure 2B) [60]. in the middle of crypts height (2nd compartment).

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Transient-amplifying cells finally differentiate into ab- was activated in a doxycycline-inducible manner, in sorptive cells — enterocytes and secretory cells (goblet contrast to control mice, cell proliferation upon YAP1 cells and enteroendocrine cells), which expand from activation was observed not only in the base of crypts the top third part of the crypt and surface epithelium but extended through the whole length of crypt’s epi- to the tip of the villus or colon surface epithelium thelium [77]. The experimental inhibition of YAP1’s (3rd compartment) [67]. activity resumed differentiation of crypts’ epithelial The base of intestinal crypts makes a niche for cells. Interestingly, YAP1 activation induced intes- ISCs, where two types of stem cells can be found: tinal dysplasia and was associated with high level of cycling crypt base columnar cells (that cycle asymmet- nuclear b-catenin expression, characteristic to active rically) and quiescent (+4) cells [68]. Leucine-rich Wnt pathway [77]. repeat-containing GPCR5 (Lgr5) is a marker present on the crypt base columnar cells, that are intestinal The role of Hippo pathway in the regeneration stem cells or long-lived multipotent progenitor cells of intestinal epithelium [68, 69]. It has been shown that Lgr5 gene is a downstream target of Wnt pathway [69]. Single Lgr5+ stem The role of YAP1 in tissue regeneration was first de- cell can rebuilt an entire crypt-villus-like structure, but scribed in dextran sodium sulphate (DSS)-induced knock-out (KO) of Lgr5 gene has no detectable effect colon regeneration model. In normal tissue, YAP1 on murine intestinal development, probably since other protein is present along the entire crypt in both related markers may attain function of the Lgr protein types of cells: proliferating and post-mitotic [13]. [68, 70]. For (+4) quiescent cells there are currently Moreover, conditional YAP1 KO in the intestinal no commonly-accepted receptors, however, Bmi1 and epithelium revealed no visible defects in cell dif- Musashi-1 have recently been proposed [71, 72]. Bmi1 ferentiation, cell death, cell proliferation or cell expression has been found in distinct cells located near migration along the crypt-villus axis [13]. Five days the bottom of crypts, which comprise rarely occurring, of DSS treatment of wild-type mice resulted in in- slowly cycling cells located predominantly in the +4 testinal inflammation and slightly decreased YAP1 position [71]. It was shown that Bmi1+ cells in the protein level. However, two days after DSS with- situation of conditional Lgr5+ cell deletion, can give drawal, YAP1 protein level dramatically increased. rise to Lgr+ cells and support crypt-villus regeneration Increased YAP1 protein level was observed (by [71, 72]. On the basis of these and other findings immunohistochemistry and WB) in regenerating a model in which Lgr5+ cells mediate homeostatic crypts [13]. This study revealed the possibility that self-renewal, whereas Bmi1+ cells mediate injury-in- Hippo signalling may be constitutively activated duced regeneration has been proposed [73]. during cell and tissue regeneration, however, Physiological level of Wnt signalling controls the during normal cell homeostasis YAP1 becomes rate of a crypt proliferation. The elevated level of Wnt inactivated. Moreover, the inactivation of tumour signalling is a characteristic feature of ISC in intestinal suppressors that normally restrict YAP1 function crypts [64] whereas deregulated Wnt signalling can may lead to tumour formation and/or overgrowth lead to colorectal cancer development [64]. R-spon- of intestinal epithelium [13]. E.g., conditional Sav1 din binds to LGR4/LGR5 receptors and enhances knock-out mice exhibited enlargement of crypts in WNT signals in ISC, but tumour suppressor RNF43 both colon and small intestine with increased num- activity negatively controls WNT signals in ISC by ber of proliferating cells. Interestingly this dysplasia ubiquitinating FZD receptors [74, 75]. Furthermore, was completely diminished by loss of YAP1 [13]. Paneth cells constitutively secrete Wnt3 ligand, but The role of MST1 and MST2 kinases in the the additional source of Wnt proteins also exists in intestinal epithelium was also investigated [13, 78]. the surrounding stromal cells. Notch signalling in the Ablation of MST1/2 kinases in mouse intestinal intestinal epithelium is involved in inhibition of the epithelium caused marked expansion of stem cell secretory phenotype of the differentiating ISC [76]. compartment and loss of secretory cells throughout The relationships between Hippo pathway and small and large intestine. Decreased phosphoryla- Wnt signalling suggest a role of the Hippo pathway tion, enhanced abundance and nuclear localization in the maintenance of the intestinal epithelium ho- of YAP1 were observed as a result of MST1/2 de- meostasis. The first reports concerning the Hippo letion in intestinal epithelium with simultaneous pathway in intestinal epithelium suggested oncogenic activation of Wnt and Notch signalling [78]. These role of YAP1. It was demonstrated that endogenous results correlate with other data obtained upon ac- YAP1 is expressed in the base of crypts where the tivation of the nuclear form of YAP1 with mutated ISCs reside [77]. In the mouse model, when YAP1 LATS1 phosphorylation site [13], because both of

©Polish Society for Histochemistry and Cytochemistry Folia Histochem Cytobiol. 2015 www.fhc.viamedica.pl 10.5603/FHC.a2015.0015 Hippo pathway in colorectal cancer 113 these conditions lead to the Hippo pathway-inde- Therefore, expression studies of Hippo pathway com- pendent activation of YAP1. ponents in CRC have been reported by some groups Divergent views on the role of YAP1 in intestinal [78, 82–86], however, only Liang et al. presented com- tissue regeneration have emerged recently. It has plex data regarding collaborative expression analysis been found that in strong cooperation with Wnt of the most important Hippo genes [87]. They found signalling the Hippo pathway controls the regenera- decreased mRNA ratios of LATS1 and MST1/2 as tion stage after injury. However, cytoplasmic YAP1 well as increased mRNA levels of YAP, TAZ, TEAD restricted elevated Wnt signalling independently of and OCT4 in CRC in comparison to healthy colon [87]. AXIN-APC-GSK complex, and in some cases acted Most of the genes coding for the Hippo pathway synergistically with the destruction complex to con- proteins were shown to function in many types of trol the subcellular localization of b-catenin [79]. In cancer either as tumour suppressors or as oncogenes mouse overexpression of YAP1 gene inhibited the (Table 2). The most commonly focused gene, YAP1, Wnt-mediated intestinal regeneration and vice versa was found to be involved in tumour development and a loss of YAP1 led to hyperactive Wnt signalling and progression in different malignancies, including CRC. expansion of stem cell niche during regeneration. It Although increased level of the YAP1 protein (as- was found that YAP1 nuclear localization correlated sessed by WB) was found in most studies [83, 88–91] with active Wnt signalling whereas its cytoplasmic suggesting its oncogenic role, the under-expression was localization inhibited Wnt pathway [79]. also observed in breast [92] and colorectal cancer [79]. Control of injury-induced proliferation of cells is The study of Barry et al. showed decreased expression crucial for maintaining the proper number of cells of YAP1 in high grade tumours and stage IV [79]. in the regenerating organ. The insufficient prolifer- The authors suggested that YAP1 is a Wnt target gene ation may lead to atrophy, whereas the dysregulated which plays a role in a negative feedback loop to limit proliferation may lead to tumourigenesis [80]. For Wnt-initiated signals in CRC development [79]. this reason focusing on mechanisms involved in re- This hypothesis sheds a new light on the possible generation of such organs like intestine, that is per- role of the Hippo pathway in the Wnt-related patho- manently exposed to potentially injuring factors, may mechanisms of the CRC development. As previously provide important clues to the pathomechanisms of mentioned, AXIN destruction complex that contains uncontrolled cellular proliferation that characterizes APC protein tightly regulates b-catenin level in Wnt neoplasms, including colorectal cancer (Figure 4). pathway (Figure 2A). Common mutations in APC gene are found in most CRC cases [81, 93, 94]. These The role of Hippo pathway mutations lead to an elevated b-catenin level in the in colorectal cancer cell nucleus where it acts as co-activator of transcription factors of the TCF/LEF family. The expression Although the most common mutations in CRC involve of the TCF4 transcription factor and the activation APC gene and dysregulated b-catenin signalling, of Wnt target genes’ transcription by the b-catenin/ YAP/TAZ can contribute to these mutations [81]. /TCF4 transcription complex were documented in colonocytes [52, 95]. It was proved that b-catenin is required for the YAP1 gene expression in HCT116, an advanced CRC cell line [95]. YAP1 gene silencing in SW620 (colon adenocarcinoma) and HCT116 (metastatic CRC) cell lines resulted in positive regulation of anchorage-independent cell growth, because decreased YAP1 level reduced growth of colonies in soft agar, especially of SW620 cells. YAP1 was detected in the cytoplasm of HCT116, SW620, SW480, RKO, LS174, and HT29 CRC cell lines, which suggests the properly functioning of Hippo pathway kinase cassette [95]. Moreover, WB protein analysis of nuclear and cytoplasmic fractions from cells grown in high confluence revealed that cell density did not affect YAP1 nuclear localization [95]. The analysis of YAP1/b-catenin expression in primary and meta Figure 4. Summary of cellular functions of Hippo pathway effector proteins YAP1/TAZ in intestinal epithelium ho- static colorectal tumours revealed that out of the meostasis and tumourigenesis 36 primary tumours examined, 86% scored positively

©Polish Society for Histochemistry and Cytochemistry Folia Histochem Cytobiol. 2015 www.fhc.viamedica.pl 10.5603/FHC.a2015.0015 114 Piotr M. Wierzbicki, Agnieszka Rybarczyk for nuclear localization of b-catenin and YAP1, while with TAZ-AXL-CTGF expression [85]. Two poten- only 3% lacked nuclear expression of either protein tial therapeutic targets, ANO1 and SQLE, were also [95]. The distribution was comparable in metastatic identified: in patients with upregulated TAZ-AXL- CRC tumours, which suggested that Wnt/b-catenin CTGF expression, a decrease in expression of either signalling pathway and Hippo/YAP1 pathway con- ANO1 and SQLE or both genes was associated with verge to promote colon cancer [95]. the survival of TAZ-AXL-CTGF high grade colon cancer patients [85]. Although the identification of Expression of Hippo pathway components EMT markers can serve as potential prognostic CRC as prognostic factors in colorectal cancer factors, the most recent data suggest caution when interpreting large scale transcriptome data from Increased expression of YAP1 in CRC was found by cancer due to contamination of tumour samples with many groups [13, 46, 78, 96]. E.g., Wang et al. showed stromal cells [99]. that YAP was overexpressed in 52.5% (73/139) of In a quantitative PCR study which focused on the CRC tumours with a predominant localization in the expression of all Hippo elements in CRC, the mRNA nucleus [97]. Furthermore, YAP1 protein expression levels of MST1 and LATS2 were decreased to higher in CRC patients correlated with cyclin D levels, nodal extent in colorectal cancer tissues than in colorectal status and pTNM stage. The authors proposed that adenomas or adjacent non-tumour tissues (non-tu- YAP1 may be a prognostic factor in CRC because mour tissues > adenomas > cancer) [87]. The mRNA of the correlation between its high expression and expression levels of YAP, TAZ and TEAD1 increased shorter overall survival [97]. in colorectal cancer compared to colorectal adeno- Wang et al. also found that YAP1 and TAZ mas and non-tumour tissues (cancer > adenomas > expression levels were significantly associated with non-tumour tissues). Similar results were obtained on the lymph node status in CRC [82]. It is known from protein level by WB analysis. The clinicopathologic other clinical and histopathological studies that have analysis revealed that expression levels of MST1, been performed for 25 years in a large surgical centre LATS2 and CDX2 (Caudal type homeobox tran- that the cut-off values of lymph node ratio are strong scription factor2, marker of throphoectoderm which independent prognostic factors for CRC patients [98]. plays important roles in endoderm and intestinal Therefore, high prognostic impact of lymph node development) in patients with lymph node metastasis metastases is a background of the statement regarding were significantly lower than those in patients without the role of YAP1 and TAZ in the prognosis of CRC lymph node metastasis. Finally the mRNA levels of patients [82]. MST1, LATS2 and CDX2 gradually decreased with Other reports showed the prognostic value of the TNM stage (I > II > III > IV) and the mRNA YAP1 and TAZ transcription levels for CRC pa- levels of YAP1, TAZ and TEAD1 gradually increased tients. A positive correlation between TAZ and YAP1 with TNM (IV > III > II > I) [87]. mRNA expression ratios and their downstream target genes AXL and CTGF was found in tumour tissues Associations of Hippo pathway of 522 CRC patients [85]. In the same study it was with therapeutic strategies in CRC demonstrated that TAZ, but not YAP mRNA level can be used to predict survival of CRC patients [85]. Personalized therapy of advanced cancer was applied Increased TAZ mRNA level may not necessarily cor- as a result of molecular progress in cancer cell biology. relate with the increase of its transcriptional activity Another important reason for personalized therapy since post-translational modifications like phospho- in CRC was the occurrence of chemoresistance of tu- rylation may cause its cytoplasmic sequestration. mour cells in some patients when the traditional drug, Therefore Yuen et al. proposed to associate TAZ 5-fluorouracil (5FU) was introduced in metastatic expression with the expression of its two downstream CRC therapy [100]. Recent reports on HT29 CRC genes, AXL and CTGF. Patients whose tumours cell line revealed high level of YAP in 5FU resistant overexpressed one, two or all of the three genes had cells. It was found that after exposing the cells to 5FU an increasing risk for disease progression [85]. Based YAP became localized in cytoplasmic compartment on microarray technique, it was also shown that target of cells and phosphorylated causing the cells to enter genes involved in EMT, migration and invasion, colon quiescence. Increased YAP protein levels were also cancer progression, cancer signalling, angiogenesis seen in human CRC liver metastases and were cor- and others factors engaged in tumourigenesis, pre- related with CRC relapse [43, 101]. sented differences in their expression level between The recently introduced biological drugs act high and low grade CRC patients which correlated directly on specific cellular targets, mainly growth

©Polish Society for Histochemistry and Cytochemistry Folia Histochem Cytobiol. 2015 www.fhc.viamedica.pl 10.5603/FHC.a2015.0015 Hippo pathway in colorectal cancer 115 factor receptors. Chemotherapy based on modern Hippo pathway, are poorly understood. The available molecular-targeting drugs like Cetuximab, Regorafenib experimental and clinical data strongly suggest that or Panitumumab (humanized monoclonal antibodies the role of the Hippo pathway should be further ex- against EGFR, CEGFR2-TIE2 and EGFR, respec- plored under different pathological conditions such as tively) showed some efficacy in advanced CRC, but cell/tissue injury or neoplastic transformation of intes- there is only scarce data regarding targeted CRC tinal epithelium. Moreover, the possible involvement therapy in respect to the Hippo pathway. of the Hippo pathway components in drug resistance Lee et al. assessed the possible connection between of advanced CRC should also be investigated. cetuximab-treated CRC patients and YAP1 mRNA expression; patients with CRC were divided into Acknowledgment two groups: one group with activated YAP1 in CRC (AYCC) and the second one with inactivated YAP1 in The study was supported by grant of the Polish Na- colorectal cancer (IYCC). Statistical analysis revealed tional Science Centre, No. N N402 683940. that patients with AYCC had slightly more advanced disease than had patients with IYCC (but patients References with stage IV were excluded from this analysis). In the 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, CA Cancer J Clin group of patients with stage I–III, the AYCC patients Jemal A. 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©Polish Society for Histochemistry and Cytochemistry Folia Histochem Cytobiol. 2015 www.fhc.viamedica.pl 10.5603/FHC.a2015.0015 116 Piotr M. Wierzbicki, Agnieszka Rybarczyk

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Submitted: 3 July, 2015 Accepted after reviews: 8 July, 2015 Available as AoP: 9 July, 2015

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